Screening of siRNA Nanoparticles for Delivery to Airway Epithelial Cells Using High Content Analysis

Background: Delivery of siRNA to the lungs via inhalation offers a unique opportunity to develop novel methods of treating a range of poorly treated respiratory conditions. However progress has been greatly hindered by safety and delivery issues. This study developed a high-throughput method for screening novel nanotechnologies for pulmonary siRNA delivery Methodology: Following physico-chemical analysis, the ability of PEI-PEG/siRNA nanoparticles to facilitate siRNA delivery was determined using high content analysis (HCA) in Calu-3 cells. Results obtained from HCA were validated using confocal microscopy. Finally, cytotoxicity of the PEI-PEG/siRNA particles was analysed by HCA using the Cellomics® multiparamter cytotoxicity assay. Conclusions: PEI-PEG/siRNA nanoparticles facilitated increased siRNA uptake and luciferase knockdown in Calu-3 cells compared to PEI/siRNA

Controlled release of vascular endothelial growth factor from spray-dried alginate microparticles in collagen-hydroxyapatite scaffolds for promoting vascularization and bone repair.

A major limitation with current tissue-engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor-eluting alginate microparticles (MPs) into freeze-dried, collagen-based scaffolds. A collagen-hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray-drying, producing particles of \u3c 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post-processing. These VEGF-MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty-defect and VEGF-free scaffolds. This biologically functionalized scaffold, composed entirely of natural-based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley \u26 Sons, Ltd

Poly(ethylene glycol)-Based Peptidomimetic “PEGtide” of Oligo-Arginine allows for efficient siRNA Transfection and gene inhibition

While a wide range of experimental and commercial transfection reagents are currently available, persistent problems remain regarding their suitability for continued development. These include the transfection efficiency for difficult-to-transfect cell types and the risks of decreased cell viability that may arise from any transfection that does occur. Therefore, research is now turning toward alternative molecules that improve the toxicity profile of the gene delivery vector (GDV), while maintaining the transfection efficiency. Among them, cell-penetrating peptides, such as octa-arginine, have shown significant potential as GDVs. Their pharmacokinetic and pharmacodynamic properties can be enhanced through peptidomimetic conversion, whereby a peptide is modified into a synthetic analogue that mimics its structure and/or function, but whose backbone is not solely based on α-amino acids. Using this technology, novel peptidomimetics were developed by co- and postpolymerization functionalization of substituted ethylene oxides, producing poly(ethylene glycol) (PEG)-based peptidomimetics termed “PEGtides”. Specifically, a PEGtide of the poly(α-amino acid) oligo-arginine [poly(glycidylguanidine)] was assessed for its ability to complex and deliver a small interfering ribonucleic acid (siRNA) using a range of cell assays and high-content analysis. PEGtide–siRNA demonstrated significantly increased internalization and gene inhibition over 24 h in Calu-3 pulmonary epithelial cells compared to commercial controls and octa-arginine-treated samples, with no evidence of toxicity. Furthermore, PEGtide–siRNA nanocomplexes can provide significant levels of gene inhibition in “difficult-to-transfect” mouse embryonic hypothalamic (mHypo N41) cells. Overall, the usefulness of this novel PEGtide for gene delivery was clearly demonstrated, establishing it as a promising candidate for continued translational research

Co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant Staphylococcus aureus (MRSA)

Bacterial resistance to antibiotics is widely regarded as a major public health concern with last resort MRSA treatments like vancomycin now encountering resistant strains. TFDs (Transcription Factor Decoys) are oligonucleotide copies of the DNA-binding sites for transcription factors. They bind to and sequester the targeted transcription factor, thus inhibiting transcription of many genes. By developing TFDs with sequences aimed at inhibiting transcription factors controlling the expression of highly conserved bacterial cell wall proteins, TFDs present as a potential method for inhibiting microbial growth without encountering typical resistance mechanisms. However, the efficient protection and delivery of the TFDs inside the bacterial cells is a critical step for the success of this technology. Therefore, in our study, specific TFDs against S. aureus were complexed with two different types of nanocarriers: cationic nanostructured lipid carriers (cNLCs) and chitosan-based nanoparticles (CS-NCs). These TFD-carrier nanocomplexes were characterized for size, zeta potential and TFD complexation or loading efficiency in a variety of buffers. In vitro activity of the nanocomplexes was examined alone and in combination with vancomycin, first in methicillin susceptible strains of S. aureus with the lead candidate advancing to tests against MRSA cultures. Results found that both cNLCs and chitosan-based carriers were adept at complexing and protecting TFDs in a range of physiological and microbiological buffers up to 72 hours. From initial testing, chitosan-TFD particles demonstrated no visible improvements in effect when co-administered with vancomycin. However, co-delivery of cNLC-TFD with vancomycin reduced the MIC of vancomycin by over 50% in MSSA and resulted in significant decreases in viability compared with vancomycin alone in MRSA cultures. Furthermore, these TFD-loaded particles demonstrated very low levels of cytotoxicity and haemolysis in vitro. To our knowledge, this is the first attempt at a combined antibiotic/oligonucleotide-TFD approach to combatting MRSA and, as such, highlights a new avenue of MRSA treatment combining traditional small molecules drugs and bacterial gene inhibition

Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics

Surface mannosylation of dispersion polymerisation derived nanoparticles by copper mediated click chemistry

The synthesis of spherical polymeric nanoparticles containing alkyne surface functionalities for post poly- merisation glycosylation is described. The nanoparticles were obtained by a polymerisation induced self- assembly (PISA) inspired methodology in dispersed media by Cu(0) mediated polymerisation. A water soluble poly(ethylene glycol methacrylate-stat-propargyl methacrylate), poly(PEGMA18-stat-PgMA5), macroinitiator was first synthesised and chain extended with 2-hydroxypropyl methacrylate (HPMA) in water using a copper wire catalyst. It was found that irrespective of the macroinitiator to HPMA ratio and the reaction time the desired spherical morphologies (<100 nm) were obtained while the absence other morphologies suggest a deviation from the classical PISA process due to chain termination in the nano- particle’s core. The obtained nanoparticles contained alkyne functionalities in the shell, which were suc- cessfully reacted by copper mediated click chemistry with fluoresceine azide and mannosides with hydro- phobic and hydrophilic spacers of different lengths. The obtained mannosylated nanoparticles displayed no significant cytotoxicity against human alveolar basal epithelial adenocarcinomic (A549) cells at any dose <0.5 mg mL−1. Preliminary binding studies confirm the ability of the mannosylated nanoparticles to bind to human lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). The methodology reported here is a convenient route to well-defined spherical and shell- functionalisable nanoparticles to create libraries of bio-active nanomaterials

Emerging Nanomedicine Therapies to Counter the Rise of Methicillin-Resistant Staphylococcus aureus

In a recent report, the World Health Organisation (WHO) classified antibiotic resistance as one of the greatest threats to global health, food security, and development. Methicillin-resistant Staphylococcus aureus (MRSA) remains at the core of this threat, with persistent and resilient strains detectable in up to 90% of S. aureus infections. Unfortunately, there is a lack of novel antibiotics reaching the clinic to address the significant morbidity and mortality that MRSA is responsible for. Recently, nanomedicine strategies have emerged as a promising therapy to combat the rise of MRSA. However, these approaches have been wide-ranging in design, with few attempts to compare studies across scientific and clinical disciplines. This review seeks to reconcile this discrepancy in the literature, with specific focus on the mechanisms of MRSA infection and how they can be exploited by bioactive molecules that are delivered by nanomedicines, in addition to utilisation of the nanomaterials themselves as antibacterial agents. Finally, we discuss targeting MRSA biofilms using nano-patterning technologies and comment on future opportunities and challenges for MRSA treatment using nanomedicine

The development of novel siRNA nanoparticles for delivery to pulmonary epithelial cells

Local delivery of short-interfering RNA (siRNA) to the lungs represents a promising means of treating a range of pulmonary conditions such as acute lung injury (ALI) and chronic obstructive pulmonary disorder (COPD). However, effective pulmonary delivery of siRNA remains hampered by a range of host defence mechanisms. Furthermore, pharmaceutical issues that are currently impeding pulmonary siRNA therapy include cytotoxicity, inefficient rates of delivery and low levels of post-delivery siRNA stability. One strategy to overcome these issues involves encapsulation of siRNA in nanoparticles. These can potentially facilitate intracellular delivery to target cells, overcome the mucus barrier and ensure stable delivery. Successful siRNA mediated therapy for respiratory disease is also dependent, however, on integration with an effective means of delivery to the appropriate area of the lungs. Herein, we have developed a range of gene delivery vectors (GDVs) suitable for inhalation for enhanced siRNA delivery to lung epithelial cells. A range of novel Poly(ethyleneimine)-Poly(ethylene) glycol (PEI-PEG) co-polymers, aminemodified cyclodextrin (SC12CD) and PEGylated peptidomimetic constructs (AOC95/99) were combined with siRNA to form siRNA nanoparticles. These were then characterised by developing high throughput, multi-parameter screening methods. These performed a detailed examination of siRNA uptake efficiency and cytotoxicity in Calu-3 bronchial epithelial cells. In vitro siRNA knockdown following siRNA nanoparticle delivery was assessed in undifferentiated and fully differentiated, mucus-producing Calu-3 cell culture. The effect of nébulisation on siRNA nanoparticle transfection efficiency was determined using PEILPEG(IOkDa) in an integrated in vitro device-cell culture model. Following this, the ability of PEI-LPEG(IOkDa) IL-8- siRNA nanoparticles to modulate IL-8 in vitro and in vivo were assessed following the establishment of an LPS-stimulated rat model of pulmonary inflammation. Results of these experiments found that PEGylation of PEI, particularly 10kDa linear PEG, resulted in significant (pin vivostudies, reductions in IL-8 gene expression were observed in lung epithelial cells but were lost at a protein level. Early indications were of PEGylation being associated with pro-inflammatory effects, however there was no significant increase compared to unmodified PEI and positive controls.</p

3D-extrusion printing of stable constructs composed of photoresponsive polypeptide hydrogels

The development of printable hydrogels with functional responsive crosslinking is vital to new age 3D printing materials in biomedical science. Disclosed here is a 3D printable UV responsive crosslinking system based on polypeptides incorporating glutamic acid, isoleucine and nitrobenzyl (NB) protected cysteine groups in a random and block copolymer. The hydrogel ink, encompassing the copolypeptide hydrogel carrier and 4-arm PEG-propiolate, can be readily extruded to produce mechanically stable constructs consisting of a number of geometries due to their remarkable shear-thinning ability. Exploiting the use of a catalyst free thiol-yne click chemistry between the cysteine residues and the propiolate groups upon UV curing, crosslinked hydrogel constructs with greater than 10 layers are fully stabilised and show improved stiffness which allow for them to be easily manipulated. This work can potentially offer a new crosslinking tool to explore in the field of 3D printing, providing highly stable hydrogel structures

ÔØ Å ÒÙ× Ö ÔØ The Development of Non-Viral Gene-Activated Matrices for Bone Regenera- tion Using Polyethyleneimine (PEI) and Collagen-Based Scaffolds The Development of Non-Viral Gene-Activated Matrices for Bone Regeneration Using Polyethyleneimine (PEI)

Abstract The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2µg dose) and transfection efficiencies (45 ± 3%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2µg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration